Tumor-intrinsic role of ICAM-1 in driving metastatic progression of triple-negative breast cancer through direct interaction with EGFR.

Triple-negative breast cancer (TNBC), the most aggressive subtype, presents a critical challenge due to the absence of approved targeted therapies. Hence, there is an urgent need to identify effective therapeutic targets for this condition. While epidermal growth factor receptor (EGFR) is prominently expressed in TNBC and recognized as a therapeutic target, anti-EGFR therapies have yet to gain approval for breast cancer treatment due to their associated side effects and limited efficacy. Here, we discovered that intercellular adhesion molecule-1 (ICAM-1) exhibits elevated expression levels in metastatic breast cancer and serves as a pivotal binding adaptor for EGFR activation, playing a crucial role in malignant progression. The activation of EGFR by tumor-expressed ICAM-1 initiates biased signaling within the JAK1/STAT3 pathway, consequently driving epithelial-to-mesenchymal transition and facilitating heightened metastasis without influencing tumor growth. Remarkably, ICAM-1-neutralizing antibody treatment significantly suppressed cancer metastasis in a breast cancer orthotopic xenograft mouse model. In conclusion, our identification of ICAM-1 as a novel tumor intrinsic regulator of EGFR activation offers valuable insights for the development of TNBC-specific anti-EGFR therapies.

Comprehensive analysis of prime editing outcomes in human embryonic stem cells.

Prime editing is a versatile and precise genome editing technique that can directly copy desired genetic modifications into target DNA sites without the need for donor DNA. This technique holds great promise for the analysis of gene function, disease modeling, and the correction of pathogenic mutations in clinically relevant cells such as human pluripotent stem cells (hPSCs). Here, we comprehensively tested prime editing in hPSCs by generating a doxycycline-inducible prime editing platform. Prime editing successfully induced all types of nucleotide substitutions and small insertions and deletions, similar to observations in other human cell types. Moreover, we compared prime editing and base editing for correcting a disease-related mutation in induced pluripotent stem cells derived form a patient with α 1-antitrypsin (A1AT) deficiency. Finally, whole-genome sequencing showed that, unlike the cytidine deaminase domain of cytosine base editors, the reverse transcriptase domain of a prime editor does not lead to guide RNA-independent off-target mutations in the genome. Our results demonstrate that prime editing in hPSCs has great potential for complementing previously developed CRISPR genome editing tools.

Mg2+-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage.

CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)-distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg2+ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg2+-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.

Engineering homoeologs provide a fine scale for quantitative traits in polyploid.

Numerous staple crops exhibit polyploidy and are difficult to genetically modify. However, recent advances in genome sequencing and editing have enabled polyploid genome engineering. The hexaploid black nightshade species Solanum nigrum has immense potential as a beneficial food supplement. We assembled its genome at the scaffold level. After functional annotations, we identified homoeologous gene sets, with similar sequence and expression profiles, based on comparative analyses of orthologous genes with close diploid relatives Solanum americanum and S. lycopersicum. Using CRISPR-Cas9-mediated mutagenesis, we generated various mutation combinations in homoeologous genes. Multiple mutants showed quantitative phenotypic changes based on the genotype, resulting in a broad-spectrum effect on the quantitative traits of hexaploid S. nigrum. Furthermore, we successfully improved the fruit productivity of Boranong, an orphan cultivar of S. nigrum suggesting that engineering homoeologous genes could be useful for agricultural improvement of polyploid crops.

NFAT1 and NFκB regulates expression of the common γ-chain cytokine receptor in activated T cells.

INTRODUCTION: Cytokines of the common γ chain (γc) family are critical for the development, differentiation, and survival of T lineage cells. Cytokines play key roles in immunodeficiencies, autoimmune diseases, allergies, and cancer. Although γc is considered an assistant receptor to transmit cytokine signals and is an indispensable receptor in the immune system, its regulatory mechanism is not yet well understood. OBJECTIVE: This study focused on the molecular mechanisms that γc expression in T cells is regulated under T cell receptor (TCR) stimulation. METHODS: The γc expression in TCR-stimulated T cells was determined by flow cytometry, western blot and quantitative RT-PCR. The regulatory mechanism of γc expression in activated T cells was examined by promoter-luciferase assay and chromatin immunoprecipitation assays. NFAT1 and NFκB deficient cells generated using CRISPR-Cas9 and specific inhibitors were used to examine their role in regulation of γc expression. Specific binding motif was confirmed by γc promotor mutant cells generated using CRISPR-Cas9. IL-7TgγcTg mice were used to examine regulatory role of γc in cytokine signaling. RESULTS: We found that activated T cells significantly upregulated γc expression, wherein NFAT1 and NFκB were key in transcriptional upregulation via T cell receptor stimulation. Also, we identified the functional binding site of the γc promoter and the synergistic effect of NFAT1 and NFκB in the regulation of γc expression. Increased γc expression inhibited IL-7 signaling and rescued lymphoproliferative disorder in an IL-7Tg animal model, providing novel insights into T cell homeostasis. CONCLUSION: Our results indicate functional cooperation between NFAT1 and NFκB in upregulating γc expression in activated T cells. As γc expression also regulates γc cytokine responsiveness, our study suggests that γc expression should be considered as one of the regulators in γc cytokine signaling and the development of T cell immunotherapies. Video Abstract.

Therapeutic base editing and prime editing of COL7A1 mutations in recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (C7), a protein that functions in skin adherence. From 36 Korean RDEB patients, we identified a total of 69 pathogenic mutations (40 variants without recurrence), including point mutations (72.5%) and insertion/deletion mutations (27.5%). For fibroblasts from two patients (Pat1 and Pat2), we applied adenine base editors (ABEs) to correct the pathogenic mutation of COL7A1 or to bypass a premature stop codon in Pat1-derived primary fibroblasts. To expand the targeting scope, we also utilized prime editors (PEs) to correct the COL7A1 mutations in Pat1- and Pat2-derived fibroblasts. Ultimately, we found that transfer of edited patient-derived skin equivalents (i.e., RDEB keratinocytes and PE-corrected RDEB fibroblasts from the RDEB patient) into the skin of immunodeficient mice led to C7 deposition and anchoring fibril formation within the dermal-epidermal junction, suggesting that base editing and prime editing could be feasible strategies for ex vivo gene editing to treat RDEB.

In vivo gene editing via homology-independent targeted integration for adrenoleukodystrophy treatment.

Adrenoleukodystrophy (ALD) is caused by various pathogenic mutations in the X-linked ABCD1 gene, which lead to metabolically abnormal accumulations of very long-chain fatty acids in many organs. However, curative treatment of ALD has not yet been achieved. To treat ALD, we applied two different gene-editing strategies, base editing and homology-independent targeted integration (HITI), in ALD patient-derived fibroblasts. Next, we performed in vivo HITI-mediated gene editing using AAV9 vectors delivered via intravenous administration in the ALD model mice. We found that the ABCD1 mRNA level was significantly increased in HITI-treated mice, and the plasma levels of C24:0-LysoPC (lysophosphatidylcholine) and C26:0-LysoPC, sensitive diagnostic markers for ALD, were significantly reduced. These results suggest that HITI-mediated mutant gene rescue could be a promising therapeutic strategy for human ALD treatment.

Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis.

There have been many engineered Cas9 variants that were developed to minimize unintended cleavage of off-target DNAs, but detailed mechanism for the way they regulate the target specificity through DNA:RNA heteroduplexation remains poorly understood. We used single-molecule FRET assay to follow the dynamics of DNA:RNA heteroduplexation for various engineered Cas9 variants with respect to on-target and off-target DNAs. Just like wild-type Cas9, these engineered Cas9 variants exhibit a strong correlation between their conformational structure and nuclease activity. Compared with wild-type Cas9, the fraction of the cleavage-competent state dropped more rapidly with increasing base-pair mismatch, which gives rise to their enhanced target specificity. We proposed a reaction model to quantitatively analyze the degree of off-target discrimination during the successive process of R-loop expansion. We found that the critical specificity enhancement step is activated during DNA:RNA heteroduplexation for evoCas9 and HypaCas9, while it occurs in the post-heteroduplexation stage for Cas9-HF1, eCas9, and Sniper-Cas9. This study sheds new light on the conformational dynamics behind the target specificity of Cas9, which will help strengthen its rational designing principles in the future.

PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing.

Prime editing technology is capable of generating targeted insertions, deletions, and base conversions. However, the process of designing prime editing guide RNAs (pegRNAs), which contain a primer binding site and a reverse-transcription template at the 3' end, is more complex than that for the single guide RNAs used with CRISPR nucleases or base editors. Furthermore, the assessment of high-throughput sequencing data after prime editors (PEs) have been employed should consider the unique feature of PEs; thus, pre-existing assessment tools cannot directly be adopted for PEs. Here, we present two user-friendly web-based tools for PEs, named PE-Designer and PE-Analyzer. PE-Designer, a dedicated tool for pegRNA selection, provides all possible target sequences, pegRNA extension sequences, and nicking guide RNA sequences together with useful information, and displays the results in an interactive image. PE-Analyzer, a dedicated tool for PE outcome analysis, accepts high-throughput sequencing data, summarizes mutation-related information in a table, and provides interactive graphs. PE-Analyzer was mainly written using JavaScript so that it can analyze several data sets without requiring that huge sequencing data (>100MB) be uploaded to the server, reducing analysis time and increasing personal security. PE-Designer and PE-Analyzer are freely available at http://www.rgenome.net/pe-designer/ and http://www.rgenome.net/pe-analyzer/ without a login process.

AC-motif: a DNA motif containing adenine and cytosine repeat plays a role in gene regulation.

I-motif or C4 is a four-stranded DNA structure with a protonated cytosine:cytosine base pair (C+:C) found in cytosine-rich sequences. We have found that oligodeoxynucleotides containing adenine and cytosine repeats form a stable secondary structure at a physiological pH with magnesium ion, which is similar to i-motif structure, and have named this structure 'adenine:cytosine-motif (AC-motif)'. AC-motif contains C+:C base pairs intercalated with putative A+:C base pairs between protonated adenine and cytosine. By investigation of the AC-motif present in the CDKL3 promoter (AC-motifCDKL3), one of AC-motifs found in the genome, we confirmed that AC-motifCDKL3 has a key role in regulating CDKL3 gene expression in response to magnesium. This is further supported by confirming that genome-edited mutant cell lines, lacking the AC-motif formation, lost this regulation effect. Our results verify that adenine-cytosine repeats commonly present in the genome can form a stable non-canonical secondary structure with a non-Watson-Crick base pair and have regulatory roles in cells, which expand non-canonical DNA repertoires.

LPA2 protein is involved in photosystem II assembly in Chlamydomonas reinhardtii.

Photosynthetic eukaryotes require the proper assembly of photosystem II (PSII) in order to strip electrons from water and fuel carbon fixation reactions. In Arabidopsis thaliana, one of the PSII subunits (CP43/PsbC) was suggested to be assembled into the PSII complex via its interaction with an auxiliary protein called Low PSII Accumulation 2 (LPA2). However, the original articles describing the role of LPA2 in PSII assembly have been retracted. To investigate the function of LPA2 in the model organism for green algae, Chlamydomonas reinhardtii, we generated knockout lpa2 mutants by using the CRISPR-Cas9 target-specific genome editing system. Biochemical analyses revealed the thylakoidal localization of LPA2 protein in the wild type (WT), whereas lpa2 mutants were characterized by a drastic reduction in the levels of D1, D2, CP47 and CP43 proteins. Consequently, reduced PSII supercomplex accumulation, chlorophyll content per cell, PSII quantum yield and photosynthetic oxygen evolution were measured in the lpa2 mutants, leading to the almost complete impairment of photoautotrophic growth. Pulse-chase experiments demonstrated that the absence of LPA2 protein caused reduced PSII assembly and reduced PSII turnover. Taken together, our data indicate that, in C. reinhardtii, LPA2 is required for PSII assembly and proper function.

Identification and Characterization of PSEUDO-RESPONSE REGULATOR (PRR) 1a and 1b Genes by CRISPR/Cas9-Targeted Mutagenesis in Chinese Cabbage (Brassica rapa L.).

The CRISPR/Cas9 site-directed gene-editing system offers great advantages for identifying gene function and crop improvement. The circadian clock measures and conveys day length information to control rhythmic hypocotyl growth in photoperiodic conditions, to achieve optimal fitness, but operates through largely unknown mechanisms. Here, we generated core circadian clock evening components, Brassica rapa PSEUDO-RESPONSE REGULATOR (BrPRR) 1a, 1b, and 1ab (both 1a and 1b double knockout) mutants, using CRISPR/Cas9 genome editing in Chinese cabbage, where 9-16 genetic edited lines of each mutant were obtained. The targeted deep sequencing showed that each mutant had 2-4 different mutation types at the target sites in the BrPRR1a and BrPRR1b genes. To identify the functions of BrPRR1a and 1b genes, hypocotyl length, and mRNA and protein levels of core circadian clock morning components, BrCCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and BrLHY (LATE ELONGATED HYPOCOTYL) a and b were examined under light/dark cycles and continuous light conditions. The BrPRR1a and 1ab double mutants showed longer hypocotyls, lower core circadian clock morning component mRNA and protein levels, and a shorter circadian rhythm than wildtype (WT). On the other hand, the BrPRR1b mutant was not significantly different from WT. These results suggested that two paralogous genes may not be associated with the same regulatory function in Chinese cabbage. Taken together, our results demonstrated that CRISPR/Cas9 is an efficient tool for achieving targeted genome modifications and elucidating the biological functions of circadian clock genes in B. rapa, for both breeding and improvement.

Genome Editing of Golden SNP-Carrying Lycopene Epsilon-Cyclase (LcyE) Gene Using the CRSPR-Cas9/HDR and Geminiviral Replicon System in Rice.

Lycopene epsilon-cyclase (LcyE) is a key enzyme in the carotenoid biosynthetic pathway of higher plants. Using the CRSPR/Cas9 and the geminiviral replicon, we optimized a method for targeted mutagenesis and golden SNP replacement of the LcyE gene in rice. We have exploited the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene via homology-directed repair (HDR). Mutagenesis experiments performed on the Donggin variety achieved precise modification of the LcyE loci with an efficiency of up to 90%. In HDR experiments, our target was the LcyE allele (LcyE-H523L) derived from anther culture containing a golden SNP replacement. The phenotype of the homologous recombination (HR) mutant obtained through the geminiviral replicon-based template delivery system was tangerine color, and the frequency was 1.32% of the transformed calli. In addition, the total carotenoid content of the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines was 6.8-9.6 times higher than that of the wild-type (WT) calli, respectively. The reactive oxygen species content was lower in the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines. These results indicate that efficient HDR can be achieved in the golden SNP replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits.

Transcriptome and Metabolite Profiling of Tomato SGR-Knockout Null Lines Using the CRISPR/Cas9 System.

Stay-green 1 (SGR1) protein is a critical regulator of chlorophyll degradation and senescence in plant leaves; however, the functions of tomato SGR1 remain ambiguous. Here, we generated an SGR1-knockout (KO) null line via clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9-mediated gene editing and conducted RNA sequencing and gas chromatography−tandem mass spectrometry analysis to identify the differentially expressed genes (DEGs). Solanum lycopersicum SGR1 (SlSGR1) knockout null line clearly showed a turbid brown color with significantly higher chlorophyll and carotenoid levels than those in the wild-type (WT) fruit. Differential gene expression analysis revealed 728 DEGs between WT and sgr#1-6 line, including 263 and 465 downregulated and upregulated genes, respectively, with fold-change >2 and adjusted p-value < 0.05. Most of the DEGs have functions related to photosynthesis, chloroplasts, and carotenoid biosynthesis. The strong changes in pigment and carotenoid content resulted in the accumulation of key primary metabolites, such as sucrose and its derivatives (fructose, galactinol, and raffinose), glycolytic intermediates (glucose, glucose-6-phosphate, and fructose-6-phosphate), and tricarboxylic acid cycle intermediates (malate and fumarate) in the leaves and fruit of the SGR-KO null lines. Overall, the SGR1-KO null lines developed here provide new evidence for the mechanisms underlying the roles of SGR1 as well as the molecular pathways involved in photosynthesis, chloroplasts, and carotenoid biosynthesis.

A thermodynamic understanding of the salt-induced B-to-Z transition of DNA containing BZ junctions.

Z-DNA has attracted interest due to its distinctive left-handed helical structure. This non-canonical DNA structure is able to form transiently and plays an important role in cellular processes such as transcriptional regulation and DNA recombination. Alternating purine-pyrimidine sequences are well known to form Z-DNA under high-salt conditions, but the detailed mechanism of B-to-Z transition of DNA containing BZ junctions under these conditions is not well understood. Here, using single-molecule FRET and circular dichroism experiments, we studied the effect of BZ junctions on Z-DNA formation under high-salt conditions. Further thermodynamic analysis revealed that a discrepancy of different DNA substrates in the presence and absence of BZ junctions in Z-DNA formation can be attributed mainly to the competition between enthalpy and entropy. Salt-induced B-to-Z transition is entropically favored in the presence of BZ junctions and is enthalpically favored in their absence. This thermodynamic information provides a deeper understanding of Z-DNA formation of DNA containing BZ junctions.

Current Status and Challenges of DNA Base Editing Tools.

CRISPR-mediated DNA base editors, which include cytosine base editors (CBEs) and adenine base editors (ABEs), are promising tools that can induce point mutations at desired sites in a targeted manner to correct or disrupt gene expression. Their high editing efficiency, coupled with their ability to generate a targeted mutation without generating a DNA double-strand break (DSB) or requiring a donor DNA template, suggests that DNA base editors will be useful for treating genetic diseases, among other applications. However, this hope has recently been challenged by the discovery of DNA base editor shortcomings, including off-target DNA editing, the generation of bystander mutations, and promiscuous deamination effects in both DNA and RNA, which arise from the main DNA base editor constituents, a Cas nuclease variant and a deaminase. In this review, we summarize information about the DNA base editors that have been developed to date, introduce their associated potential challenges, and describe current efforts to minimize or mitigate those issues of DNA base editors.

Simultaneous targeting of duplicated genes in Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins.

KEY MESSAGE: We obtained a complete mutant line of Petunia having mutations in both F3H genes via Cas9-ribonucleoproteins delivery, which exhibited a pale purplish pink flower color. The CRISPR-Cas system is now revolutionizing agriculture by allowing researchers to generate various desired mutations in plants at will. In particular, DNA-free genome editing via Cas9-ribonucleoproteins (RNPs) delivery has many advantages in plants; it does not require codon optimization or specific promoters for expression in plant cells; furthermore, it can bypass GMO regulations in some countries. Here, we have performed site-specific mutagenesis in Petunia to engineer flower color modifications. We determined that the commercial Petunia cultivar 'Madness Midnight' has two F3H coding genes and designed one guide RNA that targets both F3H genes at once. Among 67 T0 plants regenerated from Cas9-RNP transfected protoplasts, we obtained seven mutant lines that contain mutations in either F3HA or F3HB gene and one complete mutant line having mutations in both F3H genes without any selectable markers. It is noteworthy that only the f3ha f3hb exhibited a clearly modified, pale purplish pink flower color (RHS 69D), whereas the others, including the single copy gene knock-out plants, displayed purple violet (RHS 93A) flowers similar to the wild-type Petunia. To the best of our knowledge, we demonstrated a precedent of ornamental crop engineering by DNA-free CRISPR method for the first time, which will greatly accelerate a transition from a laboratory to a farmer's field.

Transcriptomic and physiological analysis of OsCAO1 knockout lines using the CRISPR/Cas9 system in rice.

KEY MESSAGE: The altered rice leaf color based on the knockout of CAO1 gene generated using CRISPR/Cas9 technology plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced senescence in rice. Rice chlorophyllide a oxygenase (OsCAO1), identified as the chlorophyll b synthesis under light condition, plays a critical role in regulating rice plant photosynthesis. In this study, the development of edited lines with pale green leaves by knockout of OsCAO1 gene known as a chlorophyll synthesis process is reported. Eighty-one genetically edited lines out of 181 T0 plants were generated through CRISPR/Cas9 system. The edited lines have short narrow flag leaves and pale green leaves compared with wild-type 'Dongjin' plants (WT). Additionally, edited lines have lower chlorophyll b and carotenoid contents both at seedling and mature stages. A transcriptome analysis identified 580 up-regulated and 206 downregulated genes in the edited lines. The differentially expressed genes (DEGs) involved in chlorophyll biosynthesis, magnesium chelatase subunit (CHLH), and glutamate-1-semialdehyde2, 1-aminomutase (GSA) metabolism decreased significantly. Meanwhile, the gel consistency (GC) levels of rice grains, chalkiness ratios and chalkiness degrees (CD) decreased in the edited lines. Thus, knockout of OsCAO1 influenced growth period, leaf development and grain quality characters of rice. Overall, the result suggests that OsCAO1 also plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced rice senescence.

Analysis of NHEJ-Based DNA Repair after CRISPR-Mediated DNA Cleavage.

Genome editing using CRISPR-Cas9 nucleases is based on the repair of the DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed repair (HDR), non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) pathways. Among these, it is thought that the NHEJ pathway is dominant and occurs throughout a cell cycle. NHEJ-based DSB repair is known to be error-prone; however, there are few studies that delve into it deeply in endogenous genes. Here, we quantify the degree of NHEJ-based DSB repair accuracy (termed NHEJ accuracy) in human-originated cells by incorporating exogenous DNA oligonucleotides. Through an analysis of joined sequences between the exogenous DNA and the endogenous target after DSBs occur, we determined that the average value of NHEJ accuracy is approximately 75% in maximum in HEK 293T cells. In a deep analysis, we found that NHEJ accuracy is sequence-dependent and the value at the DSB end proximal to a protospacer adjacent motif (PAM) is relatively lower than that at the DSB end distal to the PAM. In addition, we observed a negative correlation between the insertion mutation ratio and the degree of NHEJ accuracy. Our findings would broaden the understanding of Cas9-mediated genome editing.

Anti-Atherogenic Effect of Stem Cell Nanovesicles Targeting Disturbed Flow Sites.

Atherosclerosis development leads to irreversible cascades, highlighting the unmet need for improved methods of early diagnosis and prevention. Disturbed flow formation is one of the earliest atherogenic events, resulting in increased endothelial permeability and subsequent monocyte recruitment. Here, a mesenchymal stem cell (MSC)-derived nanovesicle (NV) that can target disturbed flow sites with the peptide GSPREYTSYMPH (PREY) (PMSC-NVs) is presented which is selected through phage display screening of a hundred million peptides. The PMSC-NVs are effectively produced from human MSCs (hMSCs) using plasmid DNA designed to functionalize the cell membrane with PREY. The potent anti-inflammatory and pro-endothelial recovery effects are confirmed, similar to those of hMSCs, employing mouse and porcine partial carotid artery ligation models as well as a microfluidic disturbed flow model with human carotid artery-derived endothelial cells. This nanoscale platform is expected to contribute to the development of new theragnostic strategies for preventing the progression of atherosclerosis.